Cloning, sequence analysis and expression of a cDNA encoding a novel insulin-like growth factor binding protein (IGFBP-2).

Insulin-like growth factors bind with high affinity to specific binding proteins in extracellular fluids. To identify structural characteristics of IGF-binding proteins that might define their physiological roles, we determined the complete primary structure of a novel human IGF-binding protein (IGFBP-2) from a cloned cDNA. The cDNA encodes a 328 amino acid IGF-binding protein precursor which contains a 39-residue signal peptide. The mature 289 amino acid IGFBP-2 has a predicted Mr of 31,325. Chinese hamster ovary (CHO) cells stably transformed with the IGFBP-2 cDNA secreted a 36 kd protein which bound, with different affinities, IGFII and IGFI, but did not bind insulin. The predicted protein sequence of this IGF-binding protein shares extensive amino acid homology (greater than 85%) with the IGF-binding protein secreted by rat BRL-3A cells, but less than 40% homology with human IGFBP-1. Therefore IGFBP-2, and not IGFBP-1 as previously suggested, represents the human homologue of the rat BRL-BP (alpha IGFBP-2). Moreover, from alignment of the predicted protein sequences of IGFBP-1 and IGFBP-2, extensive conservation of the distribution of cysteine residues is observed. Although the overall amino acid homology shared by these proteins is not high, we suggest that they represent a family of structurally related human IGFBPs. Southern blot analysis of human DNA demonstrates that IGFBP-2 is encoded by a single-copy gene, different from that of IGFBP-1.     

Expression of a mouse metallothionein-Escherichia coli β-galactosidase fusion gene (MT-βgal) in early mouse embryos

We have microinjected DNA containing the inducible mouse metallothionein-I (MT-I) promoter, coupled to the structural gene for Escherichia coli β-galactosidase (lacZ), into the pronuclei of one-cell mouse embryos. A qualitative histochemical assay, with 5-bromo-4-chloro-3-indolylβ- -galactopyranoside (X-Gal) as a substrate, was used to detect expression of lacZ at several preimplantation stages. We observed staining indicative of exogenous β-galactosidase activity in 5–17% of DNA-injected embryos assayed at preimplantation stages after 16–24 h treatment with ZnSO₄. Thus, lacZ can be used as an indicator gene for promoter function during early mouse embryogenesis, and the incorporation of the MT-I promoter into fusion genes can be a useful means of controlling the expression of exogenous genes in preimplantation mouse embryos.     

Molecular approaches to problems in biogeochemical cycling

Summary By using molecular probe techniques in combination with activity and expression measurements, it is possible to estimate bacterial populations in nature. This information can be expooited to study a number of important environmental problems. For instance, it will be possible to study ecosystem perturbation and microbial competition, by altering an ecosystem or a laboratory model of an ecosystem, and assessing corresponding changes in key activities and populations. In addition, regulation of activities in the laboratory can be compared to the response of activities and populations in situ, to develop an understanding of the key parameters that control these processes in nature. These types of approaches are important steps for determining the role of microorganisms in geochemical cycling, in both specific habitats and on a global basis.     

Field Study Comparing Growth and Viability of a Population of Phototrophic Bacteria

The growth and viability of an anoxygenic, phototrophic bacterial community in the hypolimnion of Zaca Lake, Calif., were compared throughout the summer. The community is dominated by a single species, “Thiopedia rosea,” that inhabits the entire hypolimnion (6 to 8 m) for approximately 11 months. Suboptimal conditions in the hypolimnion (extremely low light intensity, high or low H₂S levels) result in zero or extremely low growth rates (doubling times > 1 month) for most of the population, most of the time, yet cells remain viable and capable of high specific growth rates (doubling times of 1 to 10 days) when placed under favorable conditions (higher light intensities and temperatures). We first conclude that phototrophic bacterial populations in situ may frequently exist in a viable yet nongrowing state. Second, the viability of cells is likely to be reduced with depth owing to higher concentrations of potentially toxic chemicals and to changes in the physiological state associated with the prolonged periods of darkness commonly found at the bottom of bacterial plates.     

Turnover of Extracellular DNA in Eutrophic and Oligotrophic Freshwater Environments of Southwest Florida

The turnover of extracellular DNA was investigated in oligotrophic springs of the Crystal River and the eutrophic Medard Reservoir of southwest Florida. The Medard Reservoir possessed large populations of bacterioplankton and phytoplankton (6.8 × 10⁹ cells per liter and 28.6 μg of chlorophyll a per liter, respectively), while the Crystal River springs only contained a fraction of the microbial biomass found in the Medard Reservoir. Although dissolved DNA values were greater in the Medard Reservoir, higher rates of DNA removal resulted in similar extracellular DNA turnover times in both environments (9.62 ± 3.6 h in the Crystal River and 10.5 ± 2.1 h in the Medard Reservoir). These results indicate that regardless of trophic status or microbial standing stock, extracellular DNA turns over rapidly in subtropical planktonic freshwater environments. Therefore, recombinant DNA sequences from released genetically engineered microorganisms might not be expected to survive for long periods of time in freshwater planktonic environments.     

Assignment of cathepsin E (CTSE) to human chromosome region 1q31 by in situ hybridization and analysis of somatic cell hybrids

A full length cathepsin E (CTSE) cDNA clone was used to assign the corresponding gene to human chromosome region 1q31 by in situ hybridization. Southern blot analysis of DNA from three independent human x rodent somatic cell hybrids containing X;1 translocations confirmed the assignment of the CTSE gene to the distal region of the long arm of chromosome 1.     

Cloning and expression of the toxin B gene ofClostridium difficile

Results from our cloning studies on toxin A indicated that the gene for toxin B resided approximately 1 kb upstream of the toxin A gene. Clone pCD19, which contains the 5-end of the toxin A gene and a small open reading frame, was found to contain 1.2 kb of DNA which, when subcloned, expressed a nontoxic peptide that reacted with toxin B antibodies. The rest of the toxin B gene was located on the 6.8 kb cloned fragment of plasmid pCD19L. The two fragments overlapped 0.8 kb. Lysates containing protein expressed by the 6.8 fragment were cytotoxic and lethal, and were neutralized by toxin B antibody. The two fragments were ligated to give the complete toxin B gene. The protein expressed by the complete gene was cytotoxic and lethal, and showed complete immunological identity with toxin B. Further analysis of the expressed protein and the toxin B gene confirmed our earlier findings showing that toxin B has a molecular weight of 240,000 or greater.     

Studies on new,centrally active and reversible acetylcholinesterase inhibitors

We have synthesized the tertiary amines of pyridostigmine and neostigmine, 3-pyridinol dimethylcarbamate (norpyridostigmine) and 3-dimethylaminophenol dimethylcarbamate (norneostigmine) respectively, and we have tested their abilities to cross the blood-brain barrier and inhibit mouse brainAChE activity. The in vivo inhibition of AChE activity by norpyridostigmine reaches 72% at 10 minutes which is comparable to that seen with physostigmine (73% at 10 minutes). Inhibition by norneostigmine is less effective (50% at 10 minutes) and approaches that obtained with tetrahydroaminoacridine (57% at 10 minutes). These data show that both norpyridostigmine and norneostigmine cross the blood-brain barrier and that they are effective inhibitors of mouse brain AChE activity. These drugs could be useful in the treatment of memory, impairment associated with Alzheimer's disease, and other memory disorders.     

Biotransformations of aromatic aldehydes by acetogenic bacteria

Vanillin was subject to O demethylation and supported growth of Clostridium formicoaceticum and Clostridium thermoaceticum. Vanillin was also stimulatory to the CO-dependent growth of Peptostreptococcus productus. The aldehyde substituent of vanillin was metabolized by routes which were dependent upon both the acetogen and a co-metabolizable substrate (e.g. carbon monoxide [CO]). C. formicoaceticum and C. thermoaceticum oxidized the aldehyde group of vanillin to the carboxyl level, while P. productus reduced the aldehyde group of vanillin to the alcohol level. In contrast, during CO-dependent growth, C. thermoaceticum reduced 4-hydroxybenzaldehyde to 4-hydroxybenzyl alcohol while P. productus both reduced and oxidized 4-hydroxybenzaldehyde to 4-hydroxybenzyl alcohol and 4-hydroxybenzoate, respectively. These metabolic potentials indicate aromatic aldehydes may affect the flow of reductant during acetogenesis.